Mismatch repair (MMR) improves the fidelity of DNA replication by about 1000 fold by excising mismatches in the newly replicated strand arising from mis-incorporation and DNA slippage. MSH proteins initiate MMR by binding to DNA mismatches and then interacting with MLH proteins to recruit downstream repair factors. Mutations in MSH and MLH genes confer significant increases in mutation rate. MMR factors also prevent recombination between divergent DNA sequences, and process recombination intermediates containing nonhomologous single-stranded ends. The mechanisms by which these proteins identify mismatches and signal downstream factors during DNA replication and recombination are not well understood. In addition, the role of genetic background in determining the penetrance of MMR mutations with respect to disease phenotype has not been explored in depth. This proposal is focused on understanding how MMR proteins identify mismatches and signal downstream factors during DNA replication and repair, and the role of genetic background in determining the penetrance of MMR mutations. In Aim 1 we will analyze the behavior of single MSH and MLH complexes interacting with DNA using total internal fluorescence microscopy. These studies will take advantage of a large number of MMR mutants generated previously in the lab and are aimed at distinguishing between competing models for how MSH and MLH proteins signal downstream steps in MMR. In Aim 2 we will use SNP scanner technology to examine genome-wide mutation accumulation in MMR mutants. This work will allow us to determine the actual mutation rate in a MMR-defective strain and provide information that should help cancer researchers distinguish mutations critical for transformation to a cancer state from those that occur after transformation. Aim 3 is also focused on genome stability and presents in vitro and in vivo biochemical approaches to test interactions between MMR components and the SGS1 helicase to prevent recombination between divergent DNA sequences. Aim 4 outlines experiments aimed at understanding how genome instabilities arise from genetic incompatibility in MMR. This work will provide models to explain how genetic background contributes to disease penetrance in humans. In addition it offers new tools to identify genetic interactions in DNA repair pathways, with the overall goal of understanding cancer susceptibility and the molecular pathways that function in DNA repair.